JPS6357424B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is directed to 24-hydroxy-25-dehydrovitamin D ₃ , which is a novel active vitamin D ₃ compound;
The present invention relates to a method for producing the same and a calcium regulator containing the same as an active ingredient. More specifically, the present invention has excellent pharmacological effects, that is, the effect of promoting calcium absorption from the intestinal tract and increasing the calcium concentration in the blood, and the effect of dissolving bone minerals. various diseases such as osteoporosis,
The present invention relates to 24-hydroxy-25-dehydrovitamin D ₃ , which is useful as a therapeutic or preventive drug for bone lesions such as osteomalacia, a method for producing the same, and a calcium regulator containing the same as an active ingredient. Prior art As active vitamin _D3 , 1α,25-dihydroxyvitamin _D3 , 1α,24 dihydroxyvitamin
D ₃ , 1α-hydroxy-24-oxovitamin D ₃ and the like are known. Such compounds have the ability to promote calcium absorption from the intestinal tract, and are useful as therapeutic agents for bone lesions and the like. However, 24-hydroxy-25-dehydrovitamin D ₃ , which has a hydroxyl group at the 24th position and a double bond between the 25th and 26th positions, is a new compound that has not been described in any literature.
Until now, nothing was known about its production method, pharmacological activity, etc. Purpose of the Invention The purpose of the present invention is to provide 24-hydroxy-25-dehydrovitamin D ₃ , which is a novel compound and has excellent pharmacological effects, a method for producing the same, and a calcium regulator containing the same as an active ingredient. There is a particular thing. Structure and Effects of the Invention The 24-hydroxy-25-dehydrovitamin D type ₃ provided by the present invention has the following formula [] [In the formula, R represents a hydrogen atom or a hydroxyl group. The wavy line ~ represents that the carbon atom at position 24 has an R-configuration, an S-configuration, or a mixture of R-configuration and S-configuration. ] It is expressed as . 24-Hydroxy-25-dehydrovitamin D type ₃ of the above formula [] has a hydroxyl group at the 24th position and a double bond between the 25th and 26th positions, and has a completely unknown structure. . In the above formula [], R represents a hydrogen atom or a hydroxyl group. The wavy line ~ indicates that the carbon atom at position 24 is R-configured,
Indicates S-configuration or a mixture of both.
Specific examples of the compound represented by the above formula [] include 24(R)-24-hydroxy-25-dehydrovitamin D ₃ , 24(S)-24-hydroxy-25-dehydrovitamin D ₃ , 24(R) Examples include -25-dehydro-1α,24-dihydroxyvitamin D ₃ and 24(S)-25-dehydro-1α,24-dihydroxyvitamin D ₃ . 24-Hydroxy-25-dehydrovitamin D ₃ has the following formula [] [In the formula, R ¹ represents a hydrogen atom, a hydroxyl group, or a protected hydroxyl group, and R ² represents a hydrogen atom or a protective group. ] 24-oxo-25-dehydrovitamin D ₃ represented by the following formula is reduced with a borohydride compound, and then, if necessary, subjected to a deprotection reaction. In the above formula [] which is a raw material compound, R ¹
represents a hydrogen atom, a hydroxyl group or a protected hydroxyl group. The following groups can be mentioned as protecting groups for the hydroxyl group protected here. (1) Acyl group For example, acetyl group, propanoyl group, butanoyl group, pentanoyl group, caproyl group, cyclohexanoyl group, chloroacetyl group, bromoacetyl group, benzoyl group, p-bromobenzoyl group,
p-nitrobenzoyl group, ethylbenzoyl group,
C ₁ -C ₁₂ aliphatic or aromatic carboxylic acid residues such as tolyl group or their nitro-, halogen-, or alkoxy-substituted derivatives are preferably used. Among these, particularly preferred are acetyl group, benzoyl group, propanoyl group, and the like. (2) Groups that form ether bonds with hydroxyl groups, such as trimethylsilyl group, dimethyl-t-
Trialkylsilyl group such as butyl-silyl group, 2
Examples include 2-cyclic ether groups such as -tetrahydropyranyl group and 2-tetrahydrofuranyl group. (3) Alkoxycarbonyl group Examples include methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonyl group, butoxycarbonyl group, and pentoxycarbonyl group. Among the above protecting groups, particularly preferred are acyl groups,
It is an alkoxycarbonyl group, but is not limited to these. In the above formula [], R ² represents a hydrogen atom or a protective group. Such protecting groups include the same protecting groups as mentioned above. Such raw material compounds are synthesized, for example, according to the reaction formula shown below. In this reaction formula, the synthesis of compounds 3~ is JP-A-54
Publication No. 41856 is used as a reference. Compounds 9 to 8 can be synthesized by conventional ultraviolet irradiation reactions and thermal isomerization reactions. The manufacturing method of the present invention comprises 24-
It is carried out by reducing oxo-25-dehydrovitamin D ₃ with a borohydride compound. As the boron hydride compound used here, for example, the following formula [] M (R ³ ) ₄ ^- _n BH _n ... [] [In the formula, M represents an alkali metal atom, R ³
represents a lower alkyl group or a lower alkoxy group, and m represents an integer of 1 to 4. ] Preferred examples include boron hydride compounds represented by the following. In the above formula [], M represents an alkali metal atom, such as sodium, lithium, potassium, etc. R ³ represents a lower alkyl group or a lower alkoxy group, and examples of the lower alkyl group include a methyl group, an ethyl group, a propyl group, a t-butyl group, and a sec-butyl group. Examples of the lower alkoxy group include methoxy group, ethoxy group, and propoxy group. m represents an integer from 1 to 4. Examples of such borohydride compounds include sodium borohydride, lithium borohydride, lithium triethylborohydride, lithium tri-sec-butylborohydride, sodium trimethoxyborohydride, and potassium tripropoxyborohydride. etc. are listed as preferred. Particularly preferred are sodium borohydride and lithium borohydride. Boron hydride compounds are
It is preferable to use it in a range of 0.5 to 4 times the mole. When carrying out the reaction, it is advantageous to use an organic solvent; examples of the organic solvent include alcoholic solvents such as methyl alcohol, ethyl alcohol, isopropyl alcohol, and propyl alcohol; etheric solvents such as diethyl ether and tetrahydrofuran; dimethyl Examples include organic solvents such as formamide and dimethyl sulfoxide. Dissolving or suspending the 24-oxo-25-dehydrovitamin D ₃ in such an organic solvent,
It is preferable to add the borohydride compound little by little while stirring, and to proceed with the reaction by further stirring after the addition. It is preferable to cool the reaction temperature only in the initial stage in order to suppress heat generation, and then carry out the reaction at a temperature in the range of 5 to 50°C. The reaction is usually completed in several tens of minutes to several hours. When an aprotic organic solvent is used as the organic solvent, the reaction is terminated by treating the reaction solution with water or alcohol. The product thus obtained is subjected to a deprotection reaction if its hydroxyl group is protected. The deprotection reaction may be performed after isolating and purifying the product, or may be performed subsequent to the above-mentioned ring-forming reaction.
The deprotection reaction of hydroxyl groups is a known reaction per se, and can be carried out, for example, as follows. When the protecting group is an acyl group or an alkoxycarbonyl group, it may be treated in an alkaline solution of a lower aliphatic alcohol such as methanol or ethanol, or with a metal hydride such as LiAlH ₄ in ether. The temperature may be -10°C to 50°C. When the protecting group is bonded to the oxygen atom of the hydroxyl group to form an ether group, it can be easily removed by reduction or by contacting with an acid or an alkali. Conventional methods are used to isolate and purify the target product from the reaction solution. That is, means such as concentration, extraction, recrystallization, column chromatography, high performance liquid chromatography, etc. are used. Thus, by the production method of the present invention, 24-hydroxy-25-dehydrovitamin D ₃ represented by the above formula [] can be obtained. 24-Hydroxy-25-dehydrovitamin D ₃ has the effect of regulating calcium levels in the serum, that is, promoting the ability to absorb calcium from the intestinal tract and increasing the calcium concentration in the blood, and also has the effect of dissolving bone minerals. have Therefore, the 24-hydroxy-25-dehydrovitamin of the present invention
Class _D3 is extremely useful for the treatment or prevention of various diseases caused by abnormal calcium metabolism, such as osteoporosis, osteomalacia, and bone lesions in patients with renal failure. According to the present invention, the 24-hydroxy-25-dehydrovitamin represented by the above formula []
A calcium regulator containing _D3 as an active ingredient is provided. 24-Hydroxy-25-dehydrovitamin D Class ₃ can be administered either orally or parenterally, and parenteral administration includes intramuscular, subcutaneous, intravenous, and rectal administration. Among these, oral administration is preferred. Calcium regulators containing this compound as an active ingredient include tablets, powders,
Granules, suppositories, capsules, alcohol solutions,
It is used in dosage forms such as oily solutions and aqueous suspensions. When forming into a tablet, excipients such as lactose, starch, calcium carbonate, crystalline cellulose, and silicic acid; binders such as carboxymethyl cellulose, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; sodium alginate, sodium bicarbonate; , sodium lauryl sulfate, stearic acid monoglyceride, and other disintegrants; humectants such as glycerin; adsorbents such as kaolin and colloidal silicic acid; and purified talc and boric acid powder and other lubricants. can do. Powders and granules can also be formed using the above-mentioned excipients and the like in a conventional manner. When molding into the form of a suppository, it can be molded by a conventionally known method using, for example, polyethylene glycol, cacao butter, higher alcohol, gelatin, or the like. Capsules can be obtained by forming soft capsules etc. using an oily solution of the present compound. As a solvent for the oily solution, vegetable oils such as corn oil, cottonseed oil, coconut oil, almond oil, peanut oil, fish liver oil, oily esters, etc. can be used. Alcohol solutions, oil solutions, aqueous suspensions, etc. can be obtained by known methods. Antioxidants such as ascorbic acid, butylated hydroxyanisole, hydroquinone, etc. can also be incorporated into the formulation to extend the shelf life of the compounds. The dosage of 24-hydroxy-25-dehydrovitamin D ₃ of the present invention is appropriately selected depending on the patient's age, sex, degree of disease, etc.
200ng/Kg/day, more preferably 5-40ng/Kg/
It is day. From this dosage amount, the amount of 24-hydroxy-25-dehydrovitamin D ₃ to be included in the preparation in unit dosage form is determined. Hereinafter, the present invention will be explained in more detail with reference to Examples. Reference example 1 24-oxo-5α, 8α-(4-phenyl-1,
2-urazoro) Cholest-6-en-1α, 3β
-Synthesis of diol (4-) 24-oxocholester-5,7-diene-1α,
Dissolve 6.0 g of 3β-diol in 200 ml of tetrahydrofuran-methylene chloride (1:1) and dropwise add an acetone solution of 4-phenyl-1,2,4-triazoline-3,5-dione until the red color of the reaction solution disappears. did. After stirring for 1 hour, the solvent was concentrated under reduced pressure, and the resulting crude product was applied to a silica gel column (solvent: benzene-acetone system).
24-oxo-5α,8α-(4-phenyl-1,2
7.7 g of cholest-6-ene-1α,3β-diol was obtained. The physical properties of this product were as follows. MS (m/e):

[Formula] Reference Example 2 Synthesis of 1α,3β-di(methoxymethoxy)-24-oxo-5α,8α-(4-phenyl-1,2-urazolo)cholest-6-ene (5~) 24-oxo- 5α, 8α-(4-phenyl-1,
2-urazoro) Cholest-6-en-1α, 3β-
Suspend 7.5 g of diol in 50 ml of dry methylene chloride and add 6.7 g of N,N-diisopropylethylamine.
added. The mixture was cooled to 0°C, and 4.15 g of chloromethyl methyl ether was slowly added dropwise under a nitrogen stream. 1
After a period of time, the temperature was returned to room temperature, and the reaction was continued until the starting materials disappeared by TLC. After the reaction was completed, 1N hydrochloric acid was added and extracted with ethyl acetate. After sequentially washing with an aqueous sodium hydrogen carbonate solution and saturated brine, it was dried over anhydrous sodium sulfate. The solvent was concentrated under reduced pressure, and the resulting crude product was purified using a silica gel column (solvent: benzene-acetone system) to obtain 1α,3β-di(methoxymethoxy)-24-oxo-5α,8α-(4-phenyl). -1,2-Urazoro) Cholest-6-en
I got 5.8g. The physical properties of this product were as follows. NMR (CDCl ₃ ; δ, ppm) 0.85 (3H, s), 1.00 (3H, s), 1.09 (6H, d, J=7.2Hz), 3.37 (6H, s), 4.3~5.0 (4H, m) , 6.35 (2H, ABq), 7.37 (5H, s) MS (m/e);

[Formula] Reference example 3 1α,3β-di(methoxymethoxy)-24-oxo-5α,8α-(4-phenyl-1,2-urazolo)cholesto-6-en-25-ol (6~)
Synthesis of 1α,3α-di(methoxymethoxy)-24-oxo-5α,8α-(4-phenyl-1,2-urazolo)cholesto-6ene and 3.0 g of diglyme-t-
The mixture was dissolved in 90 ml of butanol (1:1), 4.95 g of t-butoxypotassium was added, and the mixture was stirred at -20°C under an oxygen atmosphere until oxygen absorption stopped. After adding 1.16g of triphenylphosphine and stirring for a while, 1N
- Hydrochloric acid and ethyl acetate were added to separate the liquid. organic layer
After sequentially washing with 1N hydrochloric acid, aqueous sodium bicarbonate solution, and saturated brine, it was dried over anhydrous sodium sulfate. The crude product obtained by concentrating the solvent under reduced pressure was transferred to a silica gel column (solvent: benzene-acetone system).
1.91g of 1α,3β-di(methoxymethoxy)-24-oxo-5α,8α-(4-phenyl-
Cholest-6-en-25-ol was obtained. The plant value of this product was as follows. NMR (CDCl ₃ ; δ, ppm) 0.86 (3H, s), 1.00 (3H, s), 1.35 (6H, s), 3.37 (6H, s), 4.3-5.0 (4H, m), 6.37 (2H, ABq), 7.3-7.9 (5H, m) MS (m/e);

[Formula] Reference example 4 Synthesis of 1α,3β-di(methoxymethoxy)-24-oxo-5α,8α-(4-phenyl-1,2-urazolo) cholester-6.25-diene (7~) 110 mg of 1α, 3β-di(methoxymethoxy)-24
-Oxo-5α,8α-(4-phenyl-1,2-
Cholest-6-en-25-ol (urazolo) was dissolved in 10 ml of benzene, 110 mg of methyl (carboxysulfamoyl) triethylammonium hydroxide was added, and the mixture was heated under reflux for 1.5 hours under a nitrogen atmosphere. Water was added, extracted with ethyl acetate, and washed with saturated brine. After drying over anhydrous sodium sulfate, the solvent was concentrated under reduced pressure and purified using a silica gel column (solvent: benzene-ethyl acetate system). 34 mg of 1α,
3β-di(methoxymethoxy)-24-oxo-5α,
8α-(4-phenyl-1,2-urazolo) cholesta-6.25-diene was obtained. The physical properties of this product were as follows. NMR (CDCl ₃ ; δ, ppm) 0.83 (3H, s), 0.98 (3H, s), 1.87 (3H, s), 3.33 (6H, s), 4.35-5.0 (4H, m), 5.73 (1H, br, s), 5.93 (1H, br, s), 6.35 (2H, ABq), 7.3-7.9 (5H, m) MS (m/e);

[Formula] Reference example 5 Synthesis of 24-oxo-1α,3β-dihydroxycholester-5,7,25-triene ( 8 ) 1α,3β-di(methoxymethoxy)-24-oxo-5α,8α-(4 -Phenyl-1,2-urazolo)cholester-6.25-diene 180mg was dissolved in 18ml of tetrahydrofuran-methanol (1:1),
A catalytic amount of concentrated hydrochloric acid was added, and the mixture was heated and stirred at 50°C.
After reacting for 15 hours, the solvent was removed under reduced pressure, and the resulting residue was dissolved in 18 ml of s-collidine and heated under reflux for 15 minutes. After the reaction was completed, 6N-hydrochloric acid was added and extracted with ethyl acetate. After sequentially washing with 1N hydrochloric acid, aqueous sodium bicarbonate solution, and saturated brine, it was dried over anhydrous sodium sulfate. The solvent was concentrated under reduced pressure, and the resulting crude product was purified twice by silica gel thin layer chromatography (solvent system: benzene-acetone system and n-hexane-2-propanol system) to obtain 22 mg of 24- Oxo-1α,3β-dihydroxycholester-5,7,25-triene was obtained. The physical properties of this product were as follows. UV (λ ^EtOH _nax,on ): 294, 282, 271, 262 (sh), 217.5 MS (m/e): 412 (M ⁺ ) Example 1 (i) 24-oxo-1α,3β-dihydroxycholesta -Deoxygenated 22mg of 5,7,25-triene
Dissolved in 600 ml of benzene-ethanol (5:1). The resulting solution was irradiated using a 200 W Hanobia lamp surrounded by a Vycor filter for 3 minutes while stirring and controlling the temperature at 5°C. This solution was then heated to reflux for 3.5 hours. After the reaction was completed, the reaction solution was concentrated under reduced pressure at 30°C or lower. The obtained crude product was purified twice by silica gel thin layer chromatography (solvent system: benzene-acetone system and n-hexane-2-propanol system). Then, the obtained purified product
Further purification was performed by high performance liquid chromatography using a Zorbax-Sil column using 1.6% methanol/dichloromethane as the eluent, and the fraction eluted at a retention time of 17.2 minutes was collected and 24-oxo-25-dehydrocarbon -1α-hydroxyvitamin
Got _D3 . The physical properties of this product were as follows. UV (EtOH, nm): λmax264, λmin237 MS (m/e): 412 (M ⁺ ), 394, 376, 361, 269, 251, 252, 134 NMR (CDCl ₃ ; δppm) 0.54 (3H, s), 0.94 (3H, d, J = 6.4Hz), 1.87 (3H, s), 4.23 (1H, m), 4.43 (1H, m), 5.00 (1H, s), 5.33 (1H, s), 5.76 (1H , s), 5.95 (1H, s), 6.01 (1H, d, J = 11.3Hz), 6.38 (1H, d, J = 11.3Hz) (ii) 24-oxo-25-dehydro-1α-hydroxyvitamin D ₃₅₀ μg was dissolved in 1 ml of ethanol, 1 mg of NaBH ₄ was added thereto, and the mixture was reacted at room temperature for 3 hours with stirring. After the reaction, 3 ml of ethyl acetate was added, followed by 2 ml of water. Ethyl acetate extraction was performed three times to extract the reaction product.
The reaction product was transferred to a Zorbax-Sil column (4.6 x 250 mm)
High performance liquid chromatography (HPLC) was performed using 2.0% methanol-dichloromethane as the eluent. By this HPLC
24(R)-25-dehydro-1α, 24-dihydroxyvitamin D ₃ and 24(S)-25-dehydro-1α,
24-dihydroxyvitamin _D3 was isolated and purified. Example 2 Calcium absorption activity from the intestinal tract and bone mineral dissolution activity Male Wistar rats immediately after weaning were fed a vitamin D-deficient low-calcium diet (Ca, 0.0036%; P,
0.3%). 250ng of 24 to this rat
(R)-25-dehydro-1α, 24-dihydroxyvitamin D ₃ or 24(S)-25-dehydro-1α,
24-Dihydroxyvitamin D ₃ with 0.2% Triton X-
Calcium absorption from the small intestine after each hour was measured using the intestinal inversion method (Martin,
DLand DcLuca, HFAm.J.Physiol. 216 , 1351
-1359 (1969)), and bone mineral dissolution activity was measured from the increase in serum calcium content. Serum calcium level was measured using the OCPC method (Connerty, HVand
Briggs, ARAm. J. Clin. Pathol. 45 , 290―296.
(1966)). The results are shown in FIGS. 1 and 2. Example 3 Binding affinity of receptor and 25-dehydro-1α,24-dihydroxyvitamin D ₃ Receptor and 24(R)-25-dehydro-1α,24
-Dihydroxyvitamin D ₃ or 24(S)-
25-dehydro-1α, 24-dihydroxyvitamin
The binding of D ₃ was carried out by the method of Eisman et al. [Eisman, JA,
Hamstra, AJ, Kream, BEand DeLuCa,
HFArch.Biochem.Biophys. 176 , 235―243
(1976)) [Ishizuka, S., Bannai.K.,
Naruchi, T. and Hashimoto, Y. Steroids, 37 ,
33-43 (1981)]. That is, the cytosolic fraction containing the receptor (0.3 mg protein/
ml) 1ml of 10000dpm [ ³ H] 1α, 25―
(OH) ₂ D ₃ (S.A163Ci/mmol) and further various concentrations of 24(R)-25-dehydro-1α,24-dihydroxyvitamin D ₃ or 24(S)-25-dehydro-1α , 24-dihydroxyvitamin D ₃ was added and incubated at 25°C for 60 minutes. 40 after reaction
% (W/V) of polyethylene glycol 6000 to 1
ml was added, stirred thoroughly, and centrifuged at 2260 x g for 60 minutes. The radioactivity of the precipitated portion obtained was measured to determine the _amount of [ ^3H ]1α,25-(OH) _2D3 bound to the receptor.
From this measurement value, when the affinity of 1α,25-(OH) ₂ D ₃ for the receptor is 1, 24(R)-
25-dehydro-1α, 24-dihydroxyvitamin
The affinity of D ₃ is 1.21, 24(S)-25-dehydro-
The affinity of 1α,24-dihydroxyvitamin _D3 is
It was 3.15. Example 4 25-dehydro-1α,24-dihydroxyvitamin D ₃ was dissolved in coconut oil to obtain an oily solution with a concentration of 7 μg/ml. Gelatin, glycerin, ethyl paraoxybenzoate, and purified water are heated and dissolved to make a coating agent, and the above oily solution is used to continuously coat the capsules so that each capsule contains 1 μg of 25-dehydro-1α,24-dihydroxyvitamin _D3. Soft capsules were manufactured using a type soft capsule manufacturing machine.

[Brief explanation of the drawing]

Figure 1 shows the calcium absorption activity of 25-dehydro-1α,24-dihydroxyvitamin D ₃ from the intestinal tract, and Figure 2 shows the bone calcium mobilization activity.

Claims (1)

[Claims] 1. The following formula [] [In the formula, R represents a hydrogen atom or a hydroxyl group. The wavy line ~ represents that the carbon atom at position 24 has an R-configuration, an S-configuration, or a mixture of R-configuration and S-configuration. ] 24-hydroxy-25-dehydrovitamin D type ₃ represented by 2 The following formula [] [In the formula, R ¹ represents a hydrogen atom, a hydroxyl group, or a protected hydroxyl group, and R ² represents a hydrogen atom or a protective group. ] 24-oxo-25-dehydrovitamin expressed as
The following formula [], which is characterized by reducing D ₃ with a borohydride compound and then subjecting it to a deprotection reaction if necessary. [In the formula, R represents a hydrogen atom or a hydroxyl group. ] A method for producing 24-hydroxy-25-dehydrovitamin D ₃ represented by 3 24-hydroxy- represented by the above formula []
A calcium regulator containing 25-dehydrovitamin D type ₃ as an active ingredient.